Peptide/protein suspending formulations

ABSTRACT

The present invention provides improved compositions for improving the chemical and physical stability of peptides and proteins. The invention provides a liquid beneficial agent formulation containing a liquid suspension comprising at least 5% by weight beneficial agent and having a viscosity and beneficial agent size which minimizes settling of the agent in suspension over the extended delivery period.

This application is a divisional of application Ser. No. 08/475,238,filed Jun. 7, 1995.

TECHNICAL FIELD

This invention relates to stabilized, concentrated suspensionsformulations of peptides and proteins. More particularly, this inventionrelates to novel and improved compositions for providing concentrated,non-aqueous suspensions of peptides/proteins for pharmaceutical usehaving adequate chemical, physical and bioactive stability suitable forlong term delivery from a sustained release drug delivery system.

BACKGROUND ART

Proteins, as well as many other biologically active compounds, degradeover time in aqueous solution. Because of this chemical instability,protein solutions are often not suitable for use in drug deliverydevices. Carriers, in which proteins do not dissolve but rather aresuspended, can often offer improved chemical stability. Furthermore, itcan be beneficial to suspend the beneficial agent in a carrier when theagent exhibits low solubility in the desired vehicle. However,suspensions can have poor physical stability due to settling andagglomeration of the suspended beneficial agent. The problems withnon-aqueous carriers tend to be exacerbated as the concentration of theactive compound is increased.

For drug delivering implants, dosing durations of up to one year are notunusual. Beneficial agents which have low therapeutic delivery rates areprime candidates for use in implants. When the device is implanted orstored, settling of the beneficial agent in the liquid formulation canoccur. This heterogeneity can adversely effect the concentration of thebeneficial agent dispensed. Compounding this problem is the size of theimplanted beneficial agent reservoir. Implant reservoirs are generallyon the order of 25-250 μl. With this volume restriction, a formulationof high concentration (greater than or equal to 10%) and a minimumamount of suspension vehicle and other excipients is preferred.

Alpha interferon (α-IFN) is one example of a beneficial agent whichprovides a therapeutic effect at a low dose. This interferon isindicated in the treatment of chronic hepatitis because of its antiviralactivity. Prescribed therapy presently entails injections of α-IFNsolution, containing about 3.0×10⁶ IU (15 micrograms) of agent per dose,three times per week for a 4 to 6 month period. Frequent injections arerequired because of the short elimination half-life of α-IFN; most ofthe drug being completely cleared from the plasma within eight to tenhours after the injection.

U.S. Pat. Nos. 4,871,538 issued to Yim et al; 4,847,079 issued to Kwanet al; 5,081,156 issued to Yamashira et al, and European Publication No.0,281,299 issued to Yim et al describe IFN/peptide compositions withconcentrations between 10⁴ to 10⁸ IU/ml. In Kwan et al a pharmaceuticalsolution having a α-IFN concentration of 10³ to 10⁸ IU/ml is described.Yim describes a dosage range being between 10⁴ to 10⁸ IU α-IFN/ml. InYim II, an insoluble complex including α-IFN, zinc, and protamine issuspended in a phosphate buffer. Yim I, Yim II, and Kuan, however, teachthe use, in part, of an aqueous buffer in their compositions. This leadsto possible hydrolysis of the compound, leading to chemical degradationand instability. Yamashira teaches a sustained release preparation ofinterferon in a n mixture with a biodegradable carrier. IFN isincorporated at concentrations of 10³ to 10⁸ IU per 1 mg of carrier or,alternatively, each dosage form containing 10⁴ to 10⁹ IU of interferon.Furthermore, while the patents and publications described above describeconcentrations between 10⁴ to 10⁹ IU/ml, none describe concentrations onthe order of 10⁹ to 10¹¹ IU/ml.

There is a need for a novel composition comprising a nonaqueoussuspension vehicle and concentrated protein/peptide as the beneficialagent for use in implanted, sustained release devices. While it is knownin the art to achieve stable α IFN concentrations of up to 10⁸ IU/ml,this invention utilizes a novel combination whose combined effectproduces a significant and surprising improvement in the physical andchemical stability of the beneficial agent compound over otherformulations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-section of an implantable sustained release osmoticdelivery device for use in combination with the concentrated suspensionsof the present invention.

FIG. 2 is a graph illustrating the stability of a cytochrome csuspension.

FIG. 3 is a graph illustrating the stability of an α-interferonsuspension.

DESCRIPTION OF THE INVENTION

One aspect of this invention relates to preparations for stabilizingpeptides and proteins at high concentrations for extended periods oftime.

Another aspect of this invention relates to stabilized preparations ofhuman α-IFN.

Another aspect of this invention relates to stabilized preparations ofhuman α-IFN having concentrations of at least 1×10⁹ IU/ml.

Another aspect of this invention relates to stabilizing beneficial agentformulations comprising a beneficial agent having a particle size ofbetween 0.3 to 50 microns and suspension vehicle formula having aviscosity between 100 to 100,000 poise at 37° C.

The new formulations are physically stable suspensions which providechemical stability to water sensitive compounds and can be employed tostabilize high concentrations of the active compound. The carriercomponents are acceptable for use in implantable systems.

MODES FOR CARRYING OUT THE INVENTION

The concentrated beneficial agent suspensions of the present inventionprovide significantly stable concentrations over extended periods oftime, useful for sustained delivery, implant applications. Thesuspensions of this invention minimize the particle degradation due tohydrolysis and particle settling over the duration of the extendeddelivery period. These extended periods of time are between one week totwo years, preferably between three months to one year.

The sustained parenteral delivery of drugs provides many advantages.Typical sustained release implantable osmotic delivery devices aredescribed in U.S. Pat. Nos. 5,034,229; 5,057,318; and 5,110,596 whichare incorporated herein by reference. As shown in FIG. 1, these devices10 typically comprise a housing 12 including a fluid impermeable wallsection 14 and a fluid permeable wall section 16 which sections defineand surround an internal compartment 18. An exit passageway 20 is formedwithin the fluid impermeable wall section to fluidly communicate theinternal compartment 18 with the external environment. To minimizeexposure to the environmental fluids, a beneficial agent 22 is containedwithin the fluid impermeable section. An expandable-driving member 24,contained within the fluid permeable section, expands with theimbibition of fluid across the fluid permeable wall section. Typically apiston 26 separates the beneficial agent 22 from the expandable drivingmember 24. This forces the agent out through the exit passageway andinto the environment of use. The non-aqueous administration of abeneficial agent in the suspension formulation as disclosed herein canbe accomplished using implant devices of these kinds.

According to this invention, high concentrations of the beneficial agentremain suspended, and physically and chemically stable in a non-aqueoussuspension vehicle. "High concentration" is defined as the beneficialagent concentration level of at least about 0.5 wt % of the formulation,preferably at least about 5 wt % and most preferably between about 10 to70% w/w. For example, "high concentrations" of α-IFN are 10⁹ to 10¹¹ ;and for salmon calcitonin, concentrations of between 2×10⁴ IU to 2.8×10⁶IU. The beneficial agent particle size is between 0.3 to 50 microns, andpreferably about 1-10 microns in diameter. Desired particle size can beprovided typically by milling, sieving, spray drying, supercriticalfluid extraction of the particular beneficial agent selected. Typicalbeneficial agent for use in this device and composition include theinterferons and calcitonin. Other representative beneficial agents thatcan be administered include pharmacologically active peptides andproteins, anabolic hormones, growth promoting hormones, hormones relatedto the endocrine system comprising porcine growth promoting hormone,bovine growth promoting hormone, equine growth promoting hormone, ovinegrowth promoting hormone, human growth promoting hormone, growthpromoting hormones derived by extraction and concentration frompituitary and hypothalmus glands, growth promoting hormones produced byrecombinant DNA methods, bovine growth promoting hormone as described inNucleic Acid Res., Vol. 10, p 7197 (1982), ovine growth promotinghormone as described in Arch. Biochem. Biophys., Vol. 156, p 493 (1973),and porcine growth promoting hormone as described in DNA, Vol. 2, pp 37,45, (1983). The polypeptides also comprise growth hormone, somatropin,somatotropin, somatotropin analogues, modified porcine somatotropin,modified bovine somatotropin, derivatives of both porcine and bovinesomatotropin, somatomedin-C, gonadotropic releasing hormone, folliclestimulating hormone, luteinizing hormone, LH-RH, LH-RH analogs growthhormone releasing factor, gonadotropin releasing factor, insulin,colchicine, chorionic gonadotropin, oxytocin, somatotropin plus an aminoacid, vasopressin, adrenocorticotrophic hormone, epidermal growthfactor, prolactin, somatostatin, somatotropin plus a protein,cosyntropin, lypressin, polypeptides such as thyrotropin releasinghormone, thyroid stimulating hormone, secretin, pancreozymin,enkephalin, glucagon, endocrine agents secreted internally anddistributed in an animal by way of the bloodstream, and the like. Thebeneficial agents and their dosage unit amounts are known to the priorart in The Pharmacological Basis of Therapeutics, by Gilman, Goodman,Rail and Murad, 7th Ed., (1985) published by MacMillan Publishing Co.,NY; in Pharmaceuical Sciences, Remington, 17th Ed., (1985) published byMack Publishing Co., Easton, Pa., and in U.S. Pat. No. 4,526,938.Particularly preferred are beneficial agents which produce the desiredtherapeutic effect at a low delivery rate/dose, for example,proteins/peptides which require sub nanograms (programs) nanograms tomilligrams of agent.

A pharmaceutically acceptable suspension vehicle is used to suspend thesolid beneficial agent particles in the beneficial agent formulation.Non-aqueous vehicles are used to isolate the beneficial agent from waterand prevent hydrolysis or other degradation of the beneficial agentwhile in suspension. Furthermore, pharmaceutically acceptable suspensionvehicles may function as a thickening agent for the components presentin an implant. As a vehicle for transporting beneficial agents from theimplant, it provides protection against the decomposition of abeneficial agent, and it imparts physical and chemical stability tocomponents present in the formulation. The thickening agent may be usedto increase the viscosity of the formulation to prevent fluids in theimplantation environment from mixing with the implant's beneficial agentformulation. The amount of thickening agent present in the formulationis between 1% to 99.9% and preferably 5-60% depending upon the viscosityadjustment needed.

Typical non-aqueous suspension vehicles include: waxes, which have asoftening temperature at or less than body temperature; hydrogenatedvegetable oils, (e.g., peanut oil, cottonseed oil, sesame oil, castoroil, olive oil, corn oil, lodinated poppy seed oils) silicon oil, mediumchain fatty acid monoglycerides, or polyols. Of these polyols arepreferred.

Polyols suitable for suspension vehicles include such as diol, triol,polyhydric alcohol, and the like. More specific polyols comprisepolyethylene glycol (average molecular weight between 200 and 1000),propylene glycol, polyethylene glycol 1,5-pentylene glycol; 1,6-hexyleneglycol; 1,7-heptylene glycol; 1,9-nonylene glycol;1,2-dimethyl-1,6-hexylene glycol; 1,2,3-propanetriol;1,2,5-pentanetriol; 1,3,5-pentanetriol; 1,2,4-butanetriol;dipentaerythriol, and the like. In another embodiment thepharmaceutically acceptable suspension vehicle comprises glycerolmono(lower alkyl) ethers and glycerol di(lower alkyl) ethers such asglycerol 1-methyl ether; glycerol 1-ethyl ether; glycerol 1,2-dimethylether; glycerol 1,3-dimethyl ether, and the like. In another embodimentthe pharmaceutically acceptable vehicle comprises a mixture such aspropylene glycol and glycerol; and the like.

Sufficient viscosity is required to suspend the particles in the carrierthroughout the duration of the extended delivery period. Settling is afunction of the particle size and the carrier viscosity. If the durationof the delivery period is shorter, the viscosity can be lower since thetime required to be suspended is shorter. The viscosity required, forexample, can be determined by the Stokes-Einstein equation which is ameasure of how far a particle in suspension will travel ##EQU1##V=velocity of settling μ=viscosity of the carrier

g=acceleration due to gravity

P_(p) =density of particle

P_(c) =density of carrier

wherein R=the average particle radius of the beneficial agent. Theviscosity of the beneficial agent suspending formulation can be alteredby the use of thickening agents to raise the viscosity to the desiredlevel. Typical thickening agents for use in the compositions of thisinvention include suitable hydrogels such as hydroxypropyl cellulose,hydroxypropyl methyl cellulose (HPMC), sodium carboxymethyl cellulose,polyacrylic acid, poly(methyl methacrylic acid) (PMMA). Preferredhydrogels are cellulose ethers such as hydroxyalkylcellulose andhydroxyalkylalkyl-cellulose compounds. A most preferredhydroxyalkylcellulose is hydroxypropyl cellulose (HPC) and povidone(PVP). Hydroxypropyl cellulose is commercially available in a wide rangeof viscosity grades sold under the tradename Klucel™ (Hercules, Ltd.,London, England). The concentration of the hydroxyalkylcellulose isdependent upon the particular viscosity grade used and the desiredviscosity of the liquid composition. For example, where the desiredviscosity is less than about 1000 poise (cps), hydroxypropyl cellulosehaving an average molecular weight of about 60,000 daltons (i.e., KlucelEF™) can be used. Where the desired viscosity is from about 1000 toabout 2500 cps, higher viscosity grades of hydroxypropyl cellulose canbe used (i.e., Klucel LF™ and Lucel GF™). In addition to using differentviscosities of different thickening agents, using different amounts ofthe same particular thickening agent can be used to vary the viscosity.Preferably, the concentration of hydroxypropyl cellulose is from 5percent w/w and, more preferably from 5 to 20% w/w of the carrier andmost preferably between 8-18% w/w. Aluminum monostearate can be used asa thickening agent if oils are used as the carrier.

Hydroxyalkylalkylcellulose ethers are a class of water-soluble hydrogelsderived from etherification of cellulose. As used herein in reference tothis class of hydrogels, the term "alkyl" means C₁ -C₆ alkyl where alkylrefers to linear or branched chains having 1 to 6 carbon atoms, whichcan be optionally substituted as herein defined. Representative alkylgroups include methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyland the like.

Exemplary hydroxyalkylalkylcelluloses are hydroxypropylmethyl cellulose,hydroxyethylmethyl cellulose and hydroxybutylmethyl cellulose.Hydroxypropylmethyl cellulose (HPMC) is preferred. HPMC is commerciallyavailable (i.e., Aldrich Chem. Co., Ltd. Dorset, England and Dow Chem.Co., Midland, Mich., USA) in a wide range of viscosity grades. Inaddition to increasing viscosity, hydroxyalkylalkylcelluloses can serveas a stabilizing, suspending and emulsifying agent. The concentration ofhydroxyalkylalkylcellulose in a liquid composition of this invention isdependent inter alia on its intended use (i.e., stabilizer, emulsifier,viscosity-increasing agent) and its viscosity grade.

To assure the viscosity of the suspension vehicle is sufficient tomaintain the agent in suspension over the desired delivery period,thickening agents can be added to the suspension vehicle. The preferredthickening agents include povidone and hydroxypropyl cellulose. In oneembodiment, when the PEG utilized is a low molecular weight, e.g., 400,5% hydroxypropyl cellulose, having an average molecular weight of 1000,or 40-60% povidone can be used in combination with a balance ofpolyethylene glycol. If the polyethylene glycol utilized in thesuspension vehicle has a molecular weight of greater than 600, e.g. 1000molecular weight, povidone is preferably utilized as the thickeningagent.

The following examples are offered to illustrate the practice of thepresent invention and are not intended to limit the invention in anymanner.

EXAMPLE 1

A viscous carrier was prepared containing 50% PEG 400 and 50% povidone(PVP) by weight. PEG 400 (Union Carbide) was weighed into a beaker andan equal weight of povidone K29-32 (GAF) was added. The PEG and povidonewere mixed by stirring with a spatula for about 5 minutes. The blendedcarrier was allowed to sit overnight to insure complete dissolution ofthe povidone. The carrier was then deaerated in a vacuum oven (NationalAppliance Company) by drawing a vacuum and holding the carrier at 50° C.for 30 minutes.

Cytochrome c (Sigma, from horseheart) was milled in a jar mill and thenpassed through a 400 mesh screen to produce a particle diameter of lessthan 37 micron. In a beaker, 0.5566 grams of the cytochrome c was addedto 4.9970 grams of the PEG 400/povidone carrier to prepare a 10%cytochrome c suspension in 50:50 PVP:peg 400 carrier. The suspension wasthoroughly blended by mixing with a spatula for about 5 minutes. Thecytochrome c suspension was then loaded into 11 osmotic veterinaryimplants (as in FIG. 1).

The implants were tested in vitro by releasing into culture tubes filledwith deionized water. To monitor release of cytochrome c from theimplants, samples of the release media were assayed on a UVspectrophotometer (Shimadzu UV 160U) at a wavelength of 409 nm. Theimplants delivered the cytochrome c successfully over the designedduration of the implant (42 days). FIG. 2 is a graph that illustratesthe cumulative protein delivery (mg) overtime. During the later half ofthe release period, several implants were removed from to show thatthere was no settling of the cytochrome c had occurred. These implantswere sectioned and samples of the protein suspension were removed fromthe top and bottom portions of the implant. The samples of the proteinsuspension were weighed, diluted with DI water in volumetric flasks andassayed via UV a spectrophotometer. Results indicated that thecytochrome c suspension was homogeneous.

EXAMPLE 2

Standard: 20 μl of a 8.0 mg/ml standard was diluted to 160 μg/ml. EachHPLC sample was diluted by a factor of 10 into distilled water. Theoperating conditions of the HPLC were as follows:

column: POROS RH 2.1 mm×3.0 cm

Mobile phase:A; 95% H2O, 0.1% TFA, 5% ACN B: 95% ACN,5% H2O, 0.083% TFA

Gradient: 20% B to 50% B in 5 minutes

Flow: 2.0 ml/min

Detector: 280 nm @ 0.002 AUFS

IRMA Standards Working standards were prepared by diluting IRMAstandards into phosphate buffered saline (PBS) containing 0.5% BovineSerum Albumin (BSA). Samples were prepared by serially diluting byfactors of 400 for formulations and 2000 for the standard into PBScontaining 0.5% BSA.

FIG. 3 shows the results of the HPLC and the IRMA assays. The HPLCmeasurements indicate no losses of the α-IFN over 5 says, even at 37° C.indicating stability of this protein non-aqueous vehicle. Relative tothe initial stock solution, the activity shown by IRMA at t=0 is 78%. Att=5 days, the formulation displayed an activity of 87% at roomtemperature and 90% at 37° C. When compared to the original stock, nolosses of α-IFN are detected by HPLC in this formulation. Stability ofinterferon in PEG over 5 days at 37° C. was indicated by this assay.However, approximately 80-90% of the activity of the initial stock wasmaintained, The IRMA readings suggest no activity losses due to time andtemperature effects.

This invention has been described in detail with particular reference tocertain preferred embodiments thereof, but it will be understood thatvariations and modifications can be effected within the spirit and scopeof the invention.

What is claimed is:
 1. A beneficial agent delivery device comprising ahousing including a fluid impermeable wall section and a fluid permeablewall section, which sections define and surround an internalcompartment, wherein the device delivers a formulation over an extendeddelivery period, and wherein the device contains a formulationcomprising:(a) at least 5% by weight beneficial agent in the form ofsolid particles having a particle size of 0.3 to 50 microns; and (b) aliquid suspension vehicle for suspending the particles in theformulation, wherein the liquid suspension formulation viscosity issufficient to prevent settling of the agent in the liquid suspensionformulation in the device over the extended delivery period.
 2. Abeneficial agent delivery device of claim 1, wherein the device isadapted to be implanted within an animal.
 3. The device of claim 1wherein the particle size is between 1 to 10 microns.
 4. The device ofclaim 1 wherein the viscosity is 100 to 100,000 poise at 37° C.
 5. Thedevice of claim 1 wherein the extended delivery period is at least about1 month.
 6. The device of claim 1 wherein the beneficial agent is awater sensitive compound.
 7. The device of claim 1 wherein thesuspension vehicle comprises a low molecular weight polyol and athickening agent.
 8. The device of claim 7 wherein the polyol ispolyethylene glycol having a molecular weight between 200 and
 1000. 9.The device of claim 8 wherein the thickening agent comprises povidone.10. The device of claim 7 wherein the polyol is polyethylene glycolhaving a molecular weight between 200 and
 600. 11. The device of claim10 wherein the thickening agent comprises povidone or hydroxypropylcellulose.
 12. The device of claim 1 wherein the beneficial agent ishuman α-interferon.
 13. The device of claim 12 wherein the concentrationof interferon is at least 1×10⁹ IU.
 14. A beneficial agent deliverydevice comprising a housing including a fluid impermeable wall sectionand a fluid permeable wall section, which sections define and surroundan internal compartment, wherein the device delivers a formulation overan extended delivery period, and wherein the device contains aformulation comprising:(a) 0.5 to 70% by weight beneficial agent in theform of solid particles having a particle size of 0.3 to 50 microns; and(b) a non-aqueous liquid vehicle comprising a non-aqueous vehicle forsuspending the particles in the formulation, polyethylene glycol with amolecular weight of 200 to 1000, and a thickening agent, which ischaracterized by a formulation viscosity of 100 to 100,000 poise at 37°C. wherein said formulation viscosity is sufficient to prevent settlingof the agent in the suspension formulation in the delivery device overthe extended delivery period.
 15. A beneficial agent delivery device ofclaim 14, wherein the device is adapted to be implanted within ananimal.
 16. The device of claim 14 wherein the particle size is between1 to 10 microns.
 17. The device of claim 14 wherein the extendeddelivery period is at least about 1 month.
 18. A device according toclaim 13 wherein the thickening agent is selected from the groupconsisting of povidone and hydroxypropyl cellulose.
 19. The device ofclaim 14 wherein the beneficial agent is human α-interferon.
 20. Thedevice of claim 19 wherein the concentration of interferon is at least1×10⁹ IU.
 21. The device of claim 14 wherein the beneficial agent is awater sensitive compound.